Structural and functional analysis of the IS903 transposase, and, The role of nucleoid host factors in transposition

The bacterial transposon IS903 is flanked at each end by perfect 18 base pair inverted repeats (IRs). The IRs delineate the transposon-host boundaries and serve as the specific binding site for the element-encoded 307 amino acid transposase. The transposase and the IRs are the minimum components required to mediate transposition. Transposition is a multi-step process that entails the movement of a DNA element from one genomic location to another. The steps of transposition are: IR binding; synaptic complex formation; cleavage of transposon DNA; target capture; and transposon insertion.; Structural aspects of the IS903 transposase as they relate to the enzyme's function were investigated using a combination of genetic and biochemical techniques. I have found that the IS903 transposase is devisable into two major domains by an extended region of protease sensitivity. The N-terminal region encompassed the first two-thirds of the protein and contained the determinants for IR-specific DNA-binding and dimerization functions, which are likely to be involved in synaptic complex formation. Residues involved in catalysis are located in both the N- and C-terminal region. Additionally, analysis of transposase cleavage products occurring in vivo showed that the protease sensitive region was a substrate for proteases in vivo . Because each domain contributes residues involved in catalysis, separation of the two domains would inactivate transposase. This possibly serves a regulatory role and contributes to the cis preference of the IS903 transposase.; Nucleoid proteins are small abundant DNA-binding proteins that have roles in organizing DNA and regulating gene expression. Several nucleoid proteins have been shown to have roles in assembly of the nucleoprotein complexes of transposition; however, there has been no systematic survey of the effect of nucleoid proteins on transposition in vivo. I have examined the effect of eliminating the six most abundantly expressed nucleoid proteins on the transposition frequency of IS903, Tn10 and Tn552. I have found that H-NS was required for efficient transposition of each of these elements, suggesting a general role for H-NS in transposition. Further analyses of target use showed that IS903 and Tn10 were affected by the absence of H-NS, suggesting a role for H-NS in target-site selection.